This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.

Nota: Il protocollo seguente descrive la SPE effettuato su 100 ml del campione d&#39;acqua con C18 sorbente e eluizione con 50% v / v di metanolo. Il campione arricchito viene diluita a raggiungere il 10% v / v di metanolo prima analisi con un enzima linked immunosorbent assay (ELISA) Kit. 1. Preparazione dei reagenti <ol><li> Preparare i campioni di acqua <ol><li> Prima di qualsiasi altro passo, filtrare ogni campione di 100 ml di acqua con filtri a 0,2 micron di dimensione dei pori. </li><li> Additiva con concentrazione desiderata diluendo il volume appropriato di soluzione di riferimento in un volume di acqua. Ad esempio, preparare 100 ml di campione con 100 ng / L di E2 diluendo 3,3 ml di soluzione di riferimento E2 ad una concentrazione di 300 mg / L. Diluire questa soluzione dieci volte per ottenere un campione a spillo con 10 ng / L E2. Diluire quest&#39;ultima altri dieci volte per ottenere un campione di 100 ml con 1 ng / L E2. </li><li> Collocare il campione filtrato (non modificato o spillo) in una bottiglia di vetro con GFilo L45. Utilizzare l&#39;esempio lo stesso giorno. Prendere una piccola frazione da analizzare con il test ELISA per stimare concentrazione iniziale. </li></ol></li><li> Preparare 300 ml di eluente diluendo in metanolo (di-) acqua deionizzata al 50% v / v in un tubo stretto. </li></ol> 2. Preparazione della colonna SPE <ol><li> Preparare a 20 mg / ml sospensione di particelle adsorbenti ottadecil-silice aggiungendo primi 1.600 ml di metanolo e poi 400 ml di acqua demineralizzata a 40 mg di sorbente in fase inversa. Strettamente chiudere il coperchio e agitare con un vortice. </li><li> Installazione della membrana inferiore della colonna <ol><li> Seleziona una membrana di nylon con dimensione dei pori 11 micron e metterlo su un doppio strato di tessuto anti-polvere. Con un diametro del punzone 3 mm, tagliare due piccole parti nella membrana. </li><li> Afferrare una delle piccole membrane con TV a fascia pinza filtra e posizionarlo su un lato della colonna. </li><li> Avvitare il connettore a fondo piatto con il tubo e stringere. La membrana è now nel luogo e l&#39;assorbente può essere aggiunto in modo sicuro. Disegnare una freccia sul corpo della colonna di puntamento verso la fine dove è stata posta la membrana. </li></ol></li><li> Preparazione della colonna assorbente imballato <ol><li> Fissare la colonna a supporto con la freccia rivolta verso il basso. La colonna deve essere impostato più verticale possibile. </li><li> Attaccare un vuoto 10 ml siringa monouso all&#39;estremità del tubo della colonna, utilizzando i connettori Luer-Lock. </li><li> Preparare un micro-pipetta con una punta di 20-200 microlitri, impostare il volume di 100 l. Questo formato puntale viene adattata alle dimensioni della colonna sorbente essere preparati. La procedura sarebbe più difficile con punte per pipette più grandi, come quelli usati con 1 ml pipette. </li><li> Agitare la sospensione sorbente con un vortice, e rapidamente pipettare 100 microlitri al centro della colonna. Mentre l&#39;iniezione, aspirare delicatamente tutta la soluzione pipettato trogolo membrana utilizzando la siringa con l&#39;altra mano. A questo stl&#39;età, la soluzione di riempimento della siringa deve essere libera da particelle, e un letto particella può eventualmente essere osservata nella colonna. </li><li> Ripetere questa operazione altre 2 volte agitando la sospensione di scorta tra tutti fasi di pipettamento per garantire la sospensione omogenea delle particelle nella soluzione. La colonna risultante contiene 6 mg di assorbente. </li><li> Quando i 300 ml di sospensione sono stati caricati ed essiccato aspirando con la siringa, tenere la siringa in posizione e posizionare la seconda membrana di nylon sulla parte superiore usando le pinze con l&#39;altra mano. </li><li> Avvitare il secondo connettore con il tubo e smaltire la siringa. La colonna SPE è pronto all&#39;uso. </li></ol></li></ol> 3. Preparazione del sistema <ol><li> Serrare colonna SPE il dispositivo utilizzando i connettori Luer-Lock. La freccia deve puntare nella stessa direzione come quella mostrata sul dispositivo. </li><li> Collegare la bottiglia contenente il campione al dispositivo avvitando ilprevisto tappo di sicurezza GL45 su di esso. </li><li> Carico 200 ml di eluente nel serbatoio &#39;Eluente&#39;. </li><li> Carico 800 ml di di-acqua nel serbatoio &#39;diluizione&#39;. </li><li> Posizionare una bottiglia all&#39;uscita rifiuti per raccogliere l&#39;acqua elaborati durante fase di estrazione. Il formato non è importante, ma il volume deve essere sufficientemente grande per quanto riguarda il volume del campione. </li><li> Mettere una piccola fiala all&#39;uscita sensore con capacità minima volume di 1,5 ml. Un volume massimo di 1 ml sarà troppo piccolo come le bolle si formano in questo sbocco al momento del ritiro eluente arricchito e tampone di diluizione. </li><li> Verificare il regolatore di pressione è in posizione chiusa ruotando manualmente, reverse-orario fino ulteriore movimento non è possibile. </li></ol> 4. SPE con il Prototype <ol><li> Accendere il prototipo premendo il pulsante sul retro. </li><li> Preparare l&#39;interfaccia utente e selezionando il programma <ol><li> Aprire l&#39;interfaccia utente. Selezionarela porta di comunicazione del computer a cui il dispositivo è collegato dalla lista, e fare clic sul pulsante &#39;Avanti&#39;. </li><li> Immettere i valori per 580 pset e 30 per dpset. La pompa regolerà per mantenere la pressione nel sistema di pressione e serbatoi a 580 ± 30 mbar durante l&#39;esecuzione. </li><li> Selezionare la modalità automatica. </li><li> Nell&#39;angolo modalità automatica, caricare il file di programma nel &#39;percorso del file di configurazione&#39; scatola. </li></ol></li><li> Regolazione del regolatore di pressione <ol><li> Avviare la pompa. </li><li> Attivare manualmente il regolatore di pressione fino a quando il valore letto per preg è inferiore ma vicino a 320 mbar. </li><li> Fermare la pompa. </li></ol></li><li> Avvio della procedura di SPE <ol><li> Premere &#39;start&#39; nell&#39;angolo modalità automatica. La pompa si accenderà e le fasi di estrazione, eluizione e diluizione seguirà automaticamente l&#39;un l&#39;altro. L&#39;intera procedura per un campione di 100 ml viene eseguita in 50 min. </li><li>Verificare il valore di preg. Deve essere compreso nell&#39;intervallo 320 - 350 mbar durante la fase di estrazione per garantire una portata ottimale. </li><li> Chiudere la piccola fiala e conservare a 4-5 ° C al buio fino al momento dell&#39;analisi. Effettuare analisi nella seguente 30 ore per impedire la degradazione dell&#39;analita. </li><li> Smaltire l&#39;acqua trattata. </li></ol></li><li> Pulizia del sistema Nota: Al termine di ogni procedura di estrazione del sistema deve essere pulito per evitare la contaminazione incrociata. <ol><li> Preparare una soluzione di 10 ml di metanolo 70% v / v in una bottiglia di vetro GL 45. </li><li> Scollegare colonna SPE e collegare il tubo di connettori. </li><li> In modalità automatica, selezionare il file &#39;pulizia&#39; e avviarlo con impostazioni di pressione stessi. </li></ol></li></ol> 5. Individuazione di Estradiolo Concentrazione con ELISA <ol><li> Preparare i campioni di calibrazione con concentrazioni come indicato nel protocollo fornito con il kit ELISA utilizzato. Preparare uno calibrazione impostato con la stessa matrice del campione, e un set di calibrazione con metanolo 10% v / v. </li><li> Dispensare la quantità necessaria di campione nei pozzetti sulla piastra, secondo le istruzioni del produttore. Utilizzare campioni di calibrazione, campioni di acqua non modificati e spillo che sono stati filtrati, ma non trattati con SPE, e arricchito i campioni in metanolo 10% v / v. Utilizzare 3 pozzetti per campione per ridurre l&#39;errore associato con il test. </li><li> Seguire le indicazioni del protocollo fornito con il kit per l&#39;aggiunta dei reagenti, tempo di incubazione, lavaggio e ulteriore reazione con l&#39;enzima. </li><li> Leggere il segnale ottico in ogni pozzetto secondo le istruzioni del produttore corredo con uno strumento lettore di piastre e raccogliere i dati. </li><li> Utilizzare il software dello strumento lettore di piastre per adattarsi alle curve di calibrazione e determinare la concentrazione di E2 nel campione iniziale con stessa matrice, e nei campioni arricchiti con metanolo 10% v / v. </li></ol>

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The authors have nothing to disclose.

È stato proposto un nuovo metodo per la preparazione dei campioni di acqua seguita da analisi mediante immunodosaggio. Lo strumento permette di eseguire l&#39;estrazione in fase solida in modo automatizzato e facile da usare. La filtrazione del campione di acqua prima della sua iniezione nel sistema è critica. Eventuali particelle ancora presenti nella soluzione potrebbe potenzialmente causare l&#39;intasamento della rete fluidica e ostacolare colonna SPE. Un altro passo importante è la p...

La preparazione del campione è un passo importante in ogni processo di analisi. In particolare, la rimozione degli effetti matrice, diminuzione delle interferenze e arricchimento dell&#39;analita sono necessari per ottenere risultati precisi e raggiungere limiti di rilevabilità. Sistema endocrino composti (IE) sono di particolare interesse per la loro azione su organismi viventi, anche quando presenti a livelli molto bassi per l&#39;ambiente. L&#39;ormone naturale 17β-estradiolo è presente sul inquinamento delle acque dell&#39;UE selezione e soggetto ad essere aggiunto alla lista delle sostanze prioritarie disciplinati dalla direttiva quadro sulle acque europee. Estrazione in fase solida (SPE) è comunemente applicato per l&#39;analisi di piccole inquinanti in acqua, sia chimica 1-5 (cromatografia, spettrometria di massa) e immunologici 6-9 metodi di rilevamento. Quest&#39;ultimo ha guadagnato interesse nel campo del monitoraggio ambientale, come saggi immunologici sono disponibili in un&#39;ampia varietà di formati, sono specifici per la taanalita rget, e raggiungere limiti di rilevabilità. 6, 7, 10, 11 Vari Enzyme Linked immunoassorbimento (ELISA) sono commercialmente disponibili e consentono di analizzare più campioni contemporaneamente su una piastra multi-pozzo. La procedura consiste in fasi successive di reazione che possono prendere un paio d&#39;ore. Il prodotto finale di reazione può essere rilevato otticamente per determinare la concentrazione della molecola bersaglio sulla base di una curva di calibrazione. Procedure SPE Classical includono sorbente pre-condizionamento, dell&#39;estrazione del campione, lavaggio, eluizione e concentrazione per evaporazione dell&#39;eluente. Il solvente utilizzato per la diluizione di questo estratto è scelto in funzione del metodo di rilevamento. Per i metodi immunologici, la quantità di solvente organico influenze fortemente la sensibilità del metodo. 12 Oltre al recupero e le prestazioni pre-concentrazione, il metodo deve anche essere semplice ed economicamente conveniente. Automazione dei SEGUIRE IL PROCEDIMENTOe aiuta a ridurre gli errori umani legati. Nel nostro lavoro precedente 13 abbiamo introdotto il nostro prototipo per SPE automatizzata, e il nostro metodo è stato applicato all&#39;analisi della ormone naturale 17β-estradiolo in campioni di acqua di mare. Con la presente il video che vorremmo evidenziare i vantaggi tecnici del nostro metodo rispetto alle tradizionali off-line e on-line SPE, e la sua particolare compatibilità con rilevamento da immuno-reazioni. Descriviamo il protocollo applicato ai campioni di acqua per la rilevazione di 17β-estradiolo. SPE viene eseguita con octadecil-silice (C18) Fase sorbente e eluizione viene eseguita con metanolo diluito.

<table border="0" cellpadding="0" cellspacing="0" width="609">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter membrane 0.2 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">GNWP04700</td>
			<td style="width:167px;">For sample filtration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon membrane 11 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">NY1104700</td>
			<td>For SPE column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable biopsy punch 5 mm</td>
			<td style="width:107px;">Medical Budget</td>
			<td style="width:119px;">39302439</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nucleodur C18 ec&#160;</td>
			<td style="width:107px;">Macherey Nagel</td>
			<td style="width:119px;">713550.01</td>
			<td>50 &#956;m particle diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic sea water</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">SSWS500-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol standard</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;"></td>
			<td>300 ng/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol ELISA kit&#160;</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;">ADI-900-008</td>
			<td>96 wells, range 30 - 3000 ng/L</td>
		</tr>
	</tbody>
</table>

a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants

Si propone un nuovo metodo per l&#39;estrazione in fase solida (SPE) di campioni di acqua ambientale. Il prototipo sviluppato è conveniente e facile da usare, e consente di eseguire un rapido, automatizzato e semplice SPE. La soluzione pre-concentrata è compatibile con l&#39;analisi mediante immunodosaggio, con un basso contenuto di solvente organico. Un metodo è descritto per l&#39;estrazione e pre-concentrazione di ormone naturale 17β-estradiolo in 100 ml campioni di acqua. Fase inversa SPE viene eseguita con sorbente ottadecil-silice e eluizione avviene con 200 ml di metanolo al 50% v / v. Eluente viene diluito con l&#39;aggiunta di acqua per abbassare la quantità di metanolo. Dopo aver preparato manualmente colonna SPE, la procedura generale viene eseguita automaticamente entro 1 ora. Al termine del processo, la concentrazione di estradiolo è misurato utilizzando un commerciale immuno-adsorbente assay enzimatico (ELISA). 100 volte pre-concentrazione si ottiene e il contenuto di metanolo in solo il 10% v / v. Recuperi completi della molecolavengono raggiunti con 1 ng / L spillo campioni deionizzata e sintetici acqua di mare.

Riproducibilità dell&#39;imballaggio sorbente è stata valutata con l&#39;essiccazione e la ponderazione del sorbente pipettato in flaconcini di vetro ed il risultato è mostrato in Figura 1. Riproducibilità del tempo di iniezione è stato testato per 100 ml di campioni, come mostrato nella Figura 2. Concentrazione iniziale e pre concentrato spillo campioni sono stati determinati utilizzando un kit ELISA commerciale pe...

Watch this Scientific Journal Video about A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants at JoVE.com

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Un metodo semplice per Automated fase solida estrazione di campioni di acqua per l&#39;analisi immunologiche di piccole inquinanti

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

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13.6K Views.  Centre Suisse d'Electronique et de Microtechnique. A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Video: A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

A Procedura piccolo volume per concentrazione virale da Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

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EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

Metodo EPA 1615. Misura di Enterovirus e Norovirus Evento in acqua per la cultura e RT-qPCR. I. Raccolta di campioni di virus

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Uso di Chironomidae (Ditteri) pupa esuvie come Rapid Bioassessment protocollo per i corpi idrici-galleggiante Surface

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Automated Gel Selection Size per migliorare la qualità delle Biblioteche di sequenziamento di nuova generazione Preparato da campioni di acqua ambientale

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

L&#39;utilizzo di una cartuccia filtrante per filtrazione di acqua campioni ed estrazione di DNA ambientale

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Protocollo per microplastiche campionamento sulla superficie del mare e Analisi dei campioni

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Un metodo di estrazione e analisi lipidica per la caratterizzazione dei microbi del suolo negli esperimenti con molti campioni

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Estrazione di pesticidi di organochlorato da pellicole di plastica e analisi di tipo plastico

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Una semplice tecnica di impianto per ristabilire gli alberi dove si verifica frequente Inundation

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.